c Recent work identified the E3 ubiquitin ligase CRL4Cdt2 as mediating the timely degradation of Cdt1 during DNA replication and following DNA damage. In both cases, proliferating cell nuclear antigen (PCNA) loaded on chromatin mediates the CRL4 Cdt2 -dependent proteolysis of Cdt1. Here, we demonstrate that while replication factor C subunit 1 (RFC1)-RFC is required for Cdt1 degradation after UV irradiation during the nucleotide excision repair process, another RFC complex, Ctf18-RFC, which is known to be involved in the establishment of cohesion, has a key role in Cdt1 degradation in S phase. Cdt1 segments having only the degron, a specific sequence element in target protein for ubiquitination, for CRL4 Cdt2 were stabilized during S phase in Ctf18-depleted cells. Additionally, endogenous Cdt1 was stabilized when both Skp2 and Ctf18 were depleted. Since a substantial amount of PCNA was detected on chromatin in Ctf18-depleted cells, Ctf18 is required in addition to loaded PCNA for Cdt1 degradation in S phase. Our data suggest that Ctf18 is involved in recruiting CRL4Cdt2 to PCNA foci during S phase. Ctf18-mediated Cdt1 proteolysis occurs independent of cohesion establishment, and depletion of Ctf18 potentiates rereplication. Our findings indicate that individual RFC complexes differentially control CRL4Cdt2 -dependent proteolysis of Cdt1 during DNA replication and repair. Maintenance of genomic information depends on faithful replication during S phase and segregation of duplicated chromosomes during mitosis, which is critical for proper cell function and survival (39). During S phase, every segment of the chromosomal DNA must be replicated only once during the cell cycle. Recent studies revealed essential roles for ubiquitin-mediated proteolysis in ensuring that DNA replication occurs only once per cell cycle (5, 7). The DNA replication-licensing factor Cdt1 associates with the origin recognition complex (ORC), which is bound to replication origins and, in conjunction with Cdc6, loads the MCM2-7 (minichromosome maintenance subunits 2 to 7) complex onto the chromatin, thereby licensing DNA for an additional round of replication. Once DNA replication is initiated upon activation of the S-phase cyclin-dependent kinases (S-CDK), relicensing of any part of the replicated regions is prevented by strict regulation of the Cdt1 protein levels in mammalian cells (38). Although Cdt1 accumulates during G 1 phase, it is degraded and maintained at low levels upon the initiation of DNA replication. S-CDK associates with Cdt1 through its Cy motif and phosphorylates Cdt1 to create a phosphorylated degron, a specific sequence element in target protein for ubiquitination, that is recognized by CRL1 Skp2 , also known as SCF Skp2 (28,37,45,51). Subsequent studies demonstrated that the Cullin4 (Cul4)-containing E3 ligase Cul4-DDB1-Cdt2, known as CRL4 Cdt2 , plays a central role in Cdt1 degradation in cells of organisms from yeast to mammals, although CRL1Skp2 operates redundantly in mammalian cells (4,17,18,20,37,44,45). The WD40 rep...

The C-terminal end of Cdt2 contains a PIP box for binding to PCNA to promote CRL4Cdt2 function, creating a new paradigm where the substrate receptor and substrates bind to a common multivalent docking platform for ubiquitination.

CRL4 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C-terminal half region of Cdt2 (Cdt2-18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2-18A was colocalized with PCNA foci during S phase in comparison with Cdt2-WT. Poly-ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2-18A. Other CRL4 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2-18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK-mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.

Background: Preventing unnecessary cell death is essential for DNA-damaged cells to carry out the DNA repair process. Results: Cdc7 inhibits the Cul4-DDB1 Cdt2 -dependent Tob degradation. Conclusion: Cdc7 enables mild DNA-damaged cells to keep their viability by competing with the Tob degradation system. Significance: Cells deal with moderate DNA damage not only by cessation of the cell cycle but also through direct mediated pro-survival signaling.

No abstract

Cdt1 is rapidly degraded by CRL4Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within »15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for »30 min in NER-deficient XP-A cells, but was degraded within »60 min. The delayed degradation was also dependent on PCNA and CRL4 Cdt2. The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

No abstract

PCNA is a DNA clamp, acting on chromatin as a platform for various proteins involved in many aspects of DNA replication-linked processes. Most of these proteins have the PCNA-interaction protein motif (PIP box) that associates with PCNA. Recent works show that PCNA plays an important role as a matchmaker, connecting PCNA-interacting proteins to the ubiquitin ligase CRL4(Cdt2) for their degradation. Proteins degraded by CRL4(Cdt2) include Cdt1, p21, and Set8 in mammalian cells. These CRL4(Cdt2) substrates have a PIP degron that consists of the canonical PIP-box sequence and additional conserved amino acids required for ubiquitination. The degradation of these proteins is triggered when PCNA is loaded onto chromatin at the onset of S phase, and this process is important to prevent re-replication of DNA. These CRL4(Cdt2) substrates are also degraded through the same mechanism in response to DNA damage. In this chapter, we describe several approaches to investigate how PIP degron-containing proteins are degraded in a PCNA-dependent manner.